1. Differential palmitoylation regulates intracellular patterning of SNAP25 Jennifer Greaves and Luke H. Chamberlain *

2. Intro SNARE proteins regulate exocytosis Syntaxin 1 and SNAP 25 well studied membrane proteins that interact w/ vesicle protein VAMP2 SNAP25 pools in recycling endosome (RE) and trans Golgi network (TGN) shuttled to and from cellular membrane Intracellular targeting thought to be regulated by palmitoylation of four cysteine sites on SNAP25

3. Palmitoylation Covalent attachment of palmitate to cysteine residue of protein Reversible + post-translational modification Mediated by palmitoyl transferases  DHHC Hydrophobic anchor…BUT also – Reg. intracellular sorting of protein – Assoc. w/ membrane microdomains – Modulate protein stability

4. Comparing fluorescent labeled SNAP25 with endogenous SNAP25 and localization in RE and TGN *PC12 cells Colocalization of eGFP-SNAP25b and WT SNAP25

5. Stained Golgi (GM130), TGN (TGN38), RE (Rab11) eGFP-SNAP25b Quantitative Colocalization Analysis to determine overlap Colocalization of eGFP-SN25b and RE, TGN, or Golgi

6. Triple Labelling Again highest association w/ RE

7. eGFP-SN25 WT vs. eGFP (85-120) Suggests palmitoylation domain is responsible for RE and TGN targeting and not interaction w/ any other SNARE proteins.

8. Addressed idea that palmitoylation only responsible for membrane anchoring Added 4CL-KrasMTD to block all cysteines so no binding of palmitate Palmitoylation mediates targeting of SNAP25 to RE and TGN membranes

9. Inhibited protein synthesis w/ Cycloheximide (CHX) to show fluorescence in RE and TGF were from dynamic palmitoylation actions of mature proteins, not newly synthesized ones.

10. Mutation of single specific palmitoylation sites Decrease in palmitate incorporation of each cysteine suggests they are All involved in palmitoylation (C88 being the least affected).

11. Mutation of single specific palmitoylation site on localization in RE and TGN All single cysteine mutations increased targeting to RE and TGN regions with C88L and C90L showing most dominant effects in directing SNAP25 localization Localization to RE + TGN is increased when Cys residues decresed from 4 to 3

12. Additional Cys incur SNAP23 activity? Fluorescence ratio = 1.0 +/- 0.022 indicating no change.

13. Cys mutant C88 did not reflect new association with Golgi No statistical analysis, difficult to tell any association with just fluorescence. But, concluded that that no colocalization of eGFP-SN25(C88L) and Golgi

14. Enhanced intracellular localization of C88 mutant affecting transportation of new proteins? Again used CHX to inhibit protein synthesis Test if C88 mutant was decreasing rate of transportation for new proteins, causing buildup of signal in intracellular targets NOT buildup, but cycling of palmitoylation affected by mutation

15. Role of Cys-rich domain directing intracellular localization of SNAP25

17. Conclusions Palmitoylation can dictate patterning of protein movement across intracellular domains. Reversibility of palmitoylation shows how changing # of palmitoylation sites (Cys) regulates localization. Cys-rich domain is “autonomous and sufficient” for intracellular localization

18. Relevence Palmitoylation’s unique dynamics to protein localization. Example of good use of controls, covering all the bases.